RESUMO
Specific and sensitive tests for the detection and typing of group A rotavirus strains are needed for a more comprehensive knowledge of the epidemiology of rotaviral infection. In this study 500 stool specimens taken from 1996 to 1998 from children with acute diarrhea in Buenos Aires were examined. Group A rotavirus was unequivocally demonstrated in 62% of the samples tested by enzyme-linked immunosorbent assay (ELISA) for detection of VP6 antigen, polyacrylamide gel electrophoresis of double-stranded RNA, and reverse transcription-PCR (RT-PCR) for amplification of the VP7:G (1, 062 bp) and VP4:P (876 bp) genes. Only five positive specimens were found by RT-PCR but not by ELISA. G and P typing was carried out by nested amplification of variable sequences of the VP7 and the VP4 genes with six G- and five P-type-specific primers (multiplex PCR). Results obtained by this method showed the prevalence of the following G and P types: G1, 39%; G2, 43%; G4, 4%; P[8], 16%; P[4], 71%. Unexpectedly, the G-P type combination most frequently found was G2P[4] (43%) rather than G1P[8] (12%), which is the most commonly found worldwide. Unusual strains of the type G1P[4] accounted for 14% of the total, while mixed infections with more than one type were found in 10% of the samples. Detection of fecal rotavirus-specific immunoglobulin M (IgM) and IgA antibodies in consecutive samples of two patients taken at daily intervals demonstrated that high levels of IgM and IgA antibodies were detected on day 1 after the onset of disease and that the samples remained positive for about 10 days, after which virus shedding was no longer observed. Multiplex PCR offers a sensitive and specific alternative to determine the prevalence of group A rotavirus G and P types and to identify the emergence of uncommon strains, whereas detection of fecal IgM and IgA antibodies represents a useful supplement to virus detection for the diagnosis of current or recently acquired infections.
Assuntos
Proteínas do Capsídeo , Capsídeo/genética , Infecções por Rotavirus/virologia , Rotavirus/classificação , Anticorpos Antivirais/análise , Antígenos Virais/análise , Argentina , Pré-Escolar , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Fezes/virologia , Genoma Viral , Genótipo , Humanos , Imunoglobulina A/análise , Imunoglobulina M/análise , Lactente , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rotavirus/genética , Rotavirus/ultraestruturaRESUMO
Specific antibodies and the activation of phagocytic cells by IFN-gamma are the key elements of the immune response involved in protection of the T. cruzi infected host. The central role of the IFN-gamma in vivo seems to be the activation of the inducible nitric oxide synthetase of macrophages (iNOS) and the production of nitric oxide (NO degree) for the intracellular destruction of the parasite. Interleukin 12 (IL-12), the cytokine that stimulates NK cells for IFN-gamma production, seems to trigger the TH1 response in the acute phase. Other cell types, such as lymphocytes Thy-1+CD4-CD8-, CD4+ and CD8+, are also involved in IFN-gamma production. The down regulation of the TH1 response could in part depend on the decrease in the macrophage activation, as a result of the controlled parasite burden, and on the production of IL-10 and transforming growth factor beta (TGF-beta). The protective TH1 immune response seems to be also related to both the tissue damage and the alterations of the immune response observed during the infection. We studied the kinetics of both NK cell activity, and the production of IL-12 and/IFN-gamma by spleen cells, as well as the seric levels of these cytokines, in BALB/c and C3H mice infected with T. cruzi, Tulahuén strain. In the spleen, we found that the production of IL-12 and the NK cell activity increased in the very early acute infection, and that in C3H the effect was higher than in BALB/c mice. IFN-gamma increased in C3H at the same time, but in the BALB/c strain it increased later in the acute phase. The infection induced a very early increase in the seric levels of IL-12, that remained high throughout the acute phase, in both mouse strains. However, the levels of IFN-gamma in the serum increased a few days before the peak of parasitemia, reaching higher values, and earlier, in BALB/c than in C3H mice. Surprisingly, in the chronic infection IL-12 production remained high in both mouse strains, but IFN-gamma production was only observed in BALB/c mice. The immune response was predominantly TH1 in both mouse strains, in spite of the higher susceptibility of BALB/c compared to C3H. The early control of the parasite burden could be evaluated as the expression of the TH1 response in spleen cells, while the seric levels of IL-12 and IFN-gamma would be related to the induction of tissue damage. Our data indicate that the protective TH1 immune response has a different expression according to the host-parasite relationship, and that the factors controlling the response are of primary importance to determine the quali- and quantitative expression of IL-12/NK/IFN-gamma as well as their involvement in resistance and tissue damage.